Abstract The global burden of HIV remains severe, and resistance develops steadily to new antiviral agents, requiring ongoing development of new therapies. Raltegravir, the first FDA-approved inhibitor of integrase (IN), binds the enzyme active site and blocks DNA strand transfer. Evolution of HIV in the presence of raltegravir elicits resistance mutants, however, motivating studies of potential additional targets on IN. IN binds the host cell protein PSIP1/LEDGF/p75 (henceforth LEDGF), which is important for efficient IN function, including targeting HIV integration to active transcription units via a tethering mechanism. We and others have found that the LEDGF binding site on IN can be bound by small molecule inhibitors (allosteric integrase inhibitors, or ALLINIs), and now highly active inhibitors targeting this site are available. Surprisingly, inhibition by these molecules does not act strongly on the integration step of the replication cycle, but instead interferes with proper maturation of the viral core after budding, implicating IN in assembly. We hypothesized that a detailed understanding of ALLINI function and escape will allow development of more potent inhibitors. We thus crystallized full-length HIV-1 IN bound to the ALLINI GSK1264, and analyzed the inhibitor interface. This represents the first time X-ray quality crystals have been generated that contained full length HIV IN. The structure shows GSK1264 bound to the dimer-interface of the catalytic domain, and also positioned at this interface is a C-terminal domain (CTD) from an adjacent IN dimer. In the crystal lattice, IN forms an open polymer mediated by this interaction. Further studies of several ALLINIs show that HIV escape mutants with reduced sensitivity commonly alter amino acids at or near the inhibitor-mediated interface, and that HIV escape mutations often encode substitutions that reduce multimerization. We propose that ALLINIs inhibit particle maturation by stimulating inappropriate polymerization of IN through the newly identified CTD-catalytic-domain interface. In preliminary data, we show that these escape mutants are far more amenable to crystallographic studies than wild-type IN, making possible aggressive new studies of IN structure and inhibition. In Specific Aim 1, we will obtain high resolution structures of IN variants in the presence of ALLINIs In Specific Aim 2, we will test our hypothesis of the mechanism of ALLINI action by generating HIV viruses carrying mutations in IN designed to disrupt the CTD-catalytic domain interface and assess their capacity for replication in cell culture and sensitivity to ALLINIs. In Specific Aim 3, we will we will take advantage of new information on IN properties to crystallize and determine the first structures of IN/DNA complexes.